Mixed-mode model made for medicinal antibodies

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  • Published: Sep 16, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: Mixed-mode model made for medicinal antibodies

Protein purification by MMC is complex

Modern biopharmaceutical production requires effective purification techniques for impure protein products, such as antibodies. Often these are purified by ion-exchange chromatography (IEC) or hydrophobic interaction chromatography (HIC). Nowadays, mixed-mode chromatography (MMC) is increasingly used. MMC resins typically purify by means of both their electrostatic and hydrophobic properties, acting as a combination of IEC and HIC.

Bispecific monoclonal antibodies are designed to bind to two different antigens, such as a tumour cell and a cytotoxic cell, at two separate binding sites. Production of such antibodies gives many unwanted impurities, some of which, such as dimers, may have similar electrostatic properties to the desired antibody and are therefore hard to separate by conventional IEC. MMC provides a possible solution to such problems. The Mannheim and Roche researchers aimed to develop a theoretical model for the purification of a bispecific antibody, a so-called ‘CrossMAb’ species, by MMC and to compare the model’s predictions with experimental results. There had previously been relatively little theoretical work carried out on this technique, which had tended to make the development of methods for such columns purely empirical.

Equations developed for MMC purification of a bispecific antibody

Equations were initially presented for the slope of the hydrophobic interaction adsorption isotherm and that of the ion exchange adsorption isotherm, relating these to various known parameters of the protein solution and column. An equation for the slope of the MMC adsorption isotherm was developed from these. The MMC column can be run either by increasing the pH at constant eluent salt concentration or by increasing the salt concentration at constant pH. An equation was deduced giving the eluting pH at fixed salt concentration, and also the eluting salt concentration at fixed pH, relating these to known parameters. Further calculations allowed theoretical chromatographic plots for protein peak elution to be produced.

Experimental runs were carried out using Capto MMC cationic resin in a GE healthcare ÄKTA Purifier 100 system. Initial purification of the antibody had left it with the dimer and the HC2LC fragment, which lacks one of the light chains, as the main impurities. The eluting buffer pH was gradually increased from 5.0 to 10.0 at various fixed salt concentrations. With increased ionic strength, the elution pH was shifted to lower values. An IEC model gave good agreement with experiment at low ionic strengths, but broke down at high ionic strengths, where the HIC effects became more important. The MMC model developed by the authors gave good agreement with experiments over the whole range of ionic strengths examined (25–500mM).

It was also possible to run the column at a fixed pH of 7.0 while increasing the buffer concentration in the eluent. Again, the theoretical results were similar to those found experimentally. Interestingly, both theory and experiment showed that this particular protein was too tightly bound at a pH of 5.0 to be eluted.

Although elution of the protein occurred in line with the theoretical predictions, clear separation from the dimer and HC2LC impurities was not achieved. The authors noted that different resin and/or eluting salts would be needed to achieve greater separation.

Theoretical approach to MMC in line with experiment

The theoretical predictions of the antibody’s elution from the MMC column were in good agreement with the experiments, showing that both IEC and HIC effects have to be taken into account when predicting elution. Such predictions are useful in process development, since they reduce development time and the need to use up valuable product, which may be in short supply in the early days of a project. It would be interesting to apply this approach to other systems to see how universal it is. The failure to clearly separate the closely related impurities under any of the experimental conditions shows how challenging antibody purification can be when such by-products are present.

Related Links

Journal of Separation Science, 2017, Early View paper. Lee et al. Modeling of bispecific antibody elution in mixed-mode cation-exchange chromatography.

Wikipedia, Bispecific Monoclonal Antibody

Wikipedia, Mixed-mode chromatography

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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